The Pathogenicity Of Haemophilus Parasuis Cytolethal Distending Toxin And Screening Of Cdt-deficient Mutant

Haemophilus parasuis, a Gram-negative bacterium, is the etiological agent of Glasser’s disease, characterized by fibrinous polyserositis, polyarthritis and meningitis. The pathogenesis, however, remains largely unknown. The cytolethal distending toxin constitutes the most1987s discovered family of bacterial toxins. CDT is composed of three subunits, and has the ability to induce DNA double strand breaks (DSBs) in eukaryotic cells, and causes irreversible cell cycle arrest or death to the target cells. The cdt gene clusters, which have the similar function with other CDT, are found in H. parasuis SH0165strain. However, there was no report about the founction of HpCDT in vivo. In view of the specific function of the toxin, while H. parasuis harm is serious and no virulence factors have been identification. Therefore, it is necessary to study the function of cdt gene cluster.The object of this study was cdtABC gene cluster of H. parasuis SH0165strain. Analyzing the function of reconstructed CDT in the infection and pathogenesis of H. parasuis by toxicity test in vitro, animal test in vivo, and construction of cdt-deficient mutant. The main research contents as follows:1. Prokaryotic expressing CDT and activity detection: According to the complete genomic sequence of H. parasuis SH0165strain in NCBI, a pair of specific primer was designed to obtain the cdt gene. Transforming into E. coli BL21(DE3) after cloning to vector pET28a, and prokaryotic expressing CDT. Porcine iliac artery endothelial cells were distension and death after incubating with CDT. The results indicate that CDT has bioactivity.2. CDT causes guinea pig tissues damage:In order to study the function of CDT in vivo, guinea pigs, which intraperitoneal injected with5mg CDT, were necropsied for evaluation of gross lesions at1,2,3,4and5d post injection. The liver and spleen were obtained to histopathological analysis. The result showed that macroscopic findings, fibin effusion, and microscopic findings, cell distension and apoptosis, were observed at3d post injection.3. CDT causes CD pig death and tissues damage:In order to analyze pathogenicity of CDT to natural animal,10mg/dose or20mg/dose was intraperitoneal injected into CD pig respectively, and the heart, liver, spleen, lung, kidney and brain were obtained to histopathological analysis. The result showed that macroscopic findings, hydropericardium, a mass of bydrothorax and ascites, fibrinous polyserositis, and microscopic findings, cell distension and apoptosis, were observed after CD pigs death caused by20mg/dose CDT. However, CD pigs, which intraperitoneal injected with10mg/dose CDT, euthanized and necropsied gross lesions were mild and just a little of fibrin clots within the peritoneal cavities. The result indicated that CDT was one of the main virulence factors of H. parasuis.4. The cdt-deficient mutant screen:According to the genomic sequence of H. parasuis SH0165strain in NCBI, a pair of specific primer was used to amplify the upstream and downstream homologous arm of cdt gene, and connected the homologous arms by overlapping PCR. The homologous arm was cloned to the temperature sensitive plasmid pSHK4TS, and transformed into H. parasuis SH0165strain. Obtained cdt gene single-exchange strain, but no cdt gene deletion mutant after screening about5000CFU. This work is going on now.Our result both in vitro and in vivo indicated that CDT is the main virulence factor that established the foundation for interpreting the pathogenesis of H. parasuis. To date, the work of cdt-deficient mutant screen is going on, which will be further interpreted the pathogenesis of H. parasuis after finishing this work.